IN SITU ALLICIN GENERATION USING TARGETED ALLIINASE DELIVERY FOR INHIBITION OF MIA PaCa 2 CELLS AND DETERMINING RELEVANT MECHANISMS RESPONSIBLE FOR INHIBITION

Abstract

Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of substrate alliin with the enzyme alliinase (EC 4.4.1.4). Allicin has been shown to be toxic to several mammalian cells in vitro in a dose-dependent manner. In the present study this cytotoxicity was taken to advantage to develop a novel approach to cancer treatment, based on site directed generation of allicin. A bacterium Cupriavidus necator with the ability of alliinase production was isolated newlinefrom soil sample and was identified by morphological, biochemical and 16S rRNA newlinesequence. Alliinase production was optimised and it was further purified to apparent homogeneity with 103-fold purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100 chromatography. The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular weight 55 kDa each. The optimum activity of the purified enzyme was found at pH 7 and the optimum temperature was 35 oC. The enzyme exhibited maximum reaction rate (Vmax) at 74.65 U/mg and Michaelis Menten constant (Km) was determined to be 0.83 mM newlinewhen alliin was used as a substrate. newline